April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Altered Protein Levels in a Human Rhodopsin-EGFP Knock-in Mouse
Author Affiliations & Notes
  • I. M. Sandoval
    Biochemistry, Baylor College of Medicine, Houston, Texas
  • A. K. Gross
    Vision Sciences, University of Alabama, Birmingham, Alabama
  • J. Mancuso
    Biochemistry, Baylor College of Medicine, Houston, Texas
  • F. Chan
    Biochemistry, Baylor College of Medicine, Houston, Texas
  • J. H. Wilson
    Biochemistry, Baylor College of Medicine, Houston, Texas
  • T. G. Wensel
    Biochemistry, Baylor College of Medicine, Houston, Texas
  • Footnotes
    Commercial Relationships  I.M. Sandoval, None; A.K. Gross, None; J. Mancuso, None; F. Chan, None; J.H. Wilson, None; T.G. Wensel, None.
  • Footnotes
    Support  NIH Grant EY1173, NIH Grant EY07981, Fellowships EY015048, DK007696 and EY007102
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5447. doi:
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      I. M. Sandoval, A. K. Gross, J. Mancuso, F. Chan, J. H. Wilson, T. G. Wensel; Altered Protein Levels in a Human Rhodopsin-EGFP Knock-in Mouse. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5447.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To determine protein and lipid levels in rod outer segments of knock-in mice with a human rhodopsin gene encoding a rhodopsin-EGFP fusion protein substituted at the mouse rhodopsin locus. Rods from these mice were previously shown to have normal photoresponse kinetics, but lowered sensitivity due to reduced effective collecting area relative to wildtype mice.

Methods: : Spectrophotometry and quantitative immunoblotting on samples of whole retinas or purified rod outer segments were used to determine protein levels. Lipid extraction and phosphate analysis were used to determine total phospholipid content.

Results: : Total rhodopsin levels (mouse rhodopsin + human rhodopsin-EGFP (hrho-EGFP) were reduced by half, and roughly 30% of the total rhodopsin was accounted for by hrho-EGFP. Transducin alpha subunit levels were reduced to 30% of wildtype levels. Levels of other phototransduction proteins, including RGS9-1, Gbeta5L, and PDE6, were unaltered. The phospholipid to rhodopsin ratio is increased in heterozygotes by 65%.

Conclusions: : Taken together with previous electrophysiological and biochemical results indicating normal photoactivation kinetics, these results suggest that G-protein activation kinetics do not depend on G-protein concentration in the physiological range. Therefore, diffusional encounter between transducin and photoactivated rhodopsin (R*) and formation of the transducin-R* complex are unlikely to determine the kinetics of phototransduction activation. Instead, a subsequent event such as the conformational change necessary for GDP-GTP exchange must be the rate limiting step of photoactivation. In addition to this surprising possibility, our findings raise two interesting hypotheses: first, the regulation of transducin expression is somehow linked to or dependent upon rhodopsin levels; second, there is a mechanism inside the cell that aims to maintain normal cell morphology in the face of alterations in rhodopsin structure and amounts.

Keywords: photoreceptors • signal transduction 

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